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Original Paper

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Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues

Wesseling M.C.a · Wagner-Britz L.a · Boukhdoud F.a · Asanidze S.a · Nguyen D.B.b · Kaestner L.c, d · Bernhardt I.a

Author affiliations

aLaboratory of Biophysics, Faculty of Natural and Technical Sciences III, Saarland University, Saarbrücken, Germany; bDepartment of Molecular Biology, Faculty of Biotechnology, Vietnam National University of Agriculture, Hanoi, Vietnam; cInstitute for Molecular Cell Biology and Rearch Centre for Molecular Imaging and Screening, School of Medicine, Saarland University, Homburg, dExperimental Physics, Saarland University, Saarbrücken, Germany

Corresponding Author

Prof. Dr. Ingolf Bernhardt

Laboratory of Biophysics, Faculty of Natural and Technical Sciences III, Saarland

University, Campus, 66123 Saarbrücken, (Germany)

Tel. +49 681 3026689, Fax +49 681 3026690, E-Mail i.bernhardt@mx.uni-saarland.de

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Cell Physiol Biochem 2016;38:2414-2425

Abstract

Background/Aims: The increase of the intracellular Ca2+ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca2+ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. Methods: The intracellular Ca2+ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 µM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca2+ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. Results: The percentage of RBCs showing increased intracellular Ca2+ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca2+ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). Conclusion: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca2+ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used.

© 2016 The Author(s) Published by S. Karger AG, Basel


Keywords

Red blood cells · Phosphatidylserine exposure · Lysophosphatidic acid · Flow cytometry · Fluorescence imaging · Ca2+ content ·


Introduction

In previous papers we have demonstrated that lysophosphatidic acid (LPA) or prostaglandin E2 (PGE2) open the non-specific, voltage-dependent cation (NSVDC) channel, which leads to an enhanced intracellular Ca2+ content of red blood cells (RBCs) [1,2], although the molecular regulation mechanism of the Ca2+ entry still remains elusive. Both substances are local mediators released from platelets after their activation within the coagulation cascade. An increase of the intracellular Ca2+ content of RBCs can be also observed after activation of protein kinase Cα (PKCα), e.g. by phorbol-12 myristate-13 acetate (PMA). Two independent Ca2+ entry processes were reported, the first is P-Type CaV2.1 channel independent and the second is associated with a likely indirect activation of CaV2.1 [3]. A higher intracellular Ca2+ content leads to an activation of the scramblase, which in turn results in a significant exposure of phosphatidylserine (PS) in the outer membrane leaflet [4,5,6]. We also demonstrated that intracellular increase in Ca2+ induced by LPA leads to cell-cell adhesion of human RBCs [7,8]. It has been reported that PS exposure in the outer membrane leaflet of the RBC membrane is of importance for the adhesion of RBCs to the endothelium in certain diseases (e.g. [9,10,11,12]). An active role of RBCs in thrombus formation has been proposed by Andrews and Low [13] whilst a correlation between decreased haematocrit and longer bleeding times has also been reported [14]. A signalling cascade was proposed by Kaestner et al. [2].

The increase of the intracellular Ca2+ content as well as the external presentation of PS after stimulation of RBCs by LPA or PMA has been investigated by many research groups [3,7,8,15,16,17,18,19,20,21,22,23]. These investigations were mainly carried out to investigate physiological parameters affecting the PS exposure. Translocation of PS from the inner to the outer membrane leaflet of RBCs is a typical sign of eryptosis (a term introduced by Lang [24]), defining the suicidal death of RBCs. A large variety of physiological parameters as well as substances have been described to induce eryptosis (e.g., [25,26,27,28,29,30,31]).

The experiments on PS exposure performed in our research group have been carried out by several investigators. During these studies we observed quantitative discrepancies with respect to different investigators but also with respect to the LPA batches used. In addition, we realized differences comparing the results of double and single labelling experiments (for Ca2+ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). We report here about the methodological approaches of the measurements of intracellular Ca2+ content as well as PS exposure to understand the challenge and to be able to compare results obtained by different research groups.

Material and Methods

Blood and solution

Human venous blood from healthy donors was obtained from the Institute of Sports and Preventive Medicine of Saarland University and the Institute of Clinical Haematology and Transfusion Medicine of Saarland University Hospital. EDTA or heparin was used as anticoagulants. Freshly drawn blood samples were stored at 4°C and used within one day. Blood was centrifuged at 2,000 g for 5 min at room temperature and the plasma and buffy coat was removed by aspiration. Subsequently, RBCs were washed 3 times in HEPES-buffered physiological solution (HPS) containing (mM): NaCl 145, KCl 7.5, glucose 10, HEPES 10, pH 7.4 under the same conditions. Finally, the RBCs were re-suspended in HPS and stored at 4°C until the beginning of the experiment. The experiment was started immediately after resuspension of the RBCs.

Single labelling experiments

Measurement of intracellular Ca2+ content: RBCs were loaded with 1 µM fluo-4 AM or 1 µM x-rhod-1 AM from a 1 mM stock solution in dimethyl sulfoxide (DMSO) in 2 ml HPS. The extracellular Ca2+ concentration was 2 mM, i.e. CaCl2 was added to the HPS. Cells were incubated at a haematocrit of 0.1 % in the dark for 30 min at 37°C with continuous shaking. Then the cells were washed again (16,000 g for 10 s), with an ice-cold HPS, re-suspended and measured as a control (at room temperature), or incubated with LPA (2.5 µM) at 37°C to activate Ca2+ uptake. Stock solutions for LPA from different companies (for comparison, 1 mM) were prepared in phosphate buffered saline. The incubation times with LPA were 1, 15 and 30 min. After incubation the cells were washed again (16,000 g for 10 s) with an ice-cold HPS, re-suspended and measured at room temperature.

Measurement of PS exposure: The cells were prepared as for measurement of the Ca2+ content. To detect the PS exposure, either annexin V-FITC or annexin V alexa fluor® 647, at a concentration of 4.5 µM was used. Annexin V binds to PS in the outer layer of the membrane and is coupled with a fluorescent dye (FITC or alexa fluor® 647), which can be measured by flow cytometry and fluorescence microscopy [32]. First a control measurement without LPA was performed. After that, LPA (2.5 µM) was added to activate the Ca2+ uptake. The cells were then incubated at 37°C for 1, 15 and 30 min. After incubation the cells were washed again (16,000 g for 10 s) with an ice-cold HPS and re-suspended. Finally, annexin V-FITC or annexin V alexa fluor® 647 was added to the cells. The staining was performed at a haematocrit of 0.1% in HPS solution with the addition of 2 mM Ca2+ at room temperature for 10 min. The measurements were also performed at room temperature.

The procedure to prepare the RBCs for measurement of intracellular Ca2+ content as well as PS exposure is based on the protocols of Nguyen et al. [15] and Wesseling et al. [16]. The time intervals between various incubation steps were kept as short as possible.

Double labelling experiments

For double labelling experiments we followed the protocols developed by Nguyen et al. [15] and Wesseling et al. [16]. The combinations of the fluorescent dyes applied were: (i) fluo-4 and annexin V alexa fluor® 647 or (ii) x-rhod-1 and annexin V-FITC to detect Ca2+ and PS, respectively.

For double labelling experiments the same procedure was used as for single labelling experiments to measure the Ca2+ content, i.e. for Ca2+ loading the extracellular Ca2+ concentration was 2 mM. The cells were stimulated with LPA and incubated at 37°C for 1, 15 and 30 min. After the last re-suspension of the RBCs for Ca2+ measurements (see above) annexin V-FITC or annexin V alexa fluor® 647 (4.5 µM) was added to detect PS and the cells incubated at room temperature for 10 min. Each sample has been measured first using flow cytometry and subsequently using fluorescence microscopy.

Flow cytometry

Intracellular free Ca2+ content as well as PS exposure was measured by flow cytometry (FACS Calibur and Cell Quest Pro software, Becton Dickinson Biosciences, Franklin Lakes, USA) as described before [15,16,33]. Ca2+ was measured in the FL-1 channel (excitation at 488 nm, emission at 520/15 nm) for fluo-4 and in the FL-2 channel (excitation at 550 nm, emission at 585/42 nm) for x-rhod-1. PS exposure in case of annexin V-FITC was detected also in the FL-1 channel. In case of annexin V alexa fluor® 647 it was detected in the FL-4 channel (excitation at 633 nm, emission at 661/16 nm) with a xenon diode. In all cases the negative and positive gates were identified based on control experiments (without stimulating substances) and A23187 (2 µM), respectively. Compensation was not necessary since there was no overlapping. The relative fluorescence intensity was analysed using the mean value of 30,000 cells from each blood sample. For each condition at least three different blood samples were used.

Fluorescence microscopy

The RBCs were monitored using an inverted fluorescence microscope (Eclipse TE2000-E, Nikon, Tokyo, Japan) as described before [15,16]. The diluted RBC samples (approximately 0.025% haematocrit) were placed on a cover slip in a dark room at room temperature. Images were taken with an electron multiplication CCD camera (CCD97, Photometrics, Tucson, USA) using a 100x1.4 (NA) oil immersion lens with infinity corrected optics. From each RBC sample, 5 images from different positions of the cover slip randomly chosen were taken using the imaging software VisiView (Visitron Systems, Puchheim, Germany). Each image consists of one transmitted light (exposure time 200 ms) and one (for single labelling) or two (for double labelling) fluorescence shot (exposure time 4 s). Fluo-4 and annexin V-FITC were excited with a xenon lamp-based monochromator (Visitron Systems, Puchheim, Germany) at a centre wavelength of 488 nm. Emission was recorded at 520/15 nm. X-rhod-1 and annexin V alexa fluor® 647 were excited at a centre wavelength of 543 nm and 661 nm, respectively. Emission was recorded at 610/20 nm for x-rhod-1 and 660/20 nm for annexin V alexa fluor® 647.

Reagents

If not mentioned otherwise, all chemicals used were purchased from Sigma-Aldrich (Munich, Germany). LPA was obtained from Sigma-Aldrich, Cayman Chemical Company (Ann Arbor, USA), and Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Fluo-4 AM and annexin V-FITC were obtained from Molecular Probes (Eugene, USA) and annexin V alexa fluor® 647 from Roche Diagnostics GmbH (Mannheim, Germany).

Statistical significance

Data are presented as mean values ± SD of at least 3 independent experiments. The significance of differences was tested by ANOVA. Statistical significance of the data was defined as follows: p > 0.05 (n.s.); 0.01 < p ≤ 0.05 (*); 0.001 < p ≤ 0.01 (**); p ≤ 0.001 (***).

Results and Discussion

Comparison of RBC activation using LPA from different companies

In our experiments described in previous papers [3,15,16,21] we realised that RBC stimulation with LPA from different batches obtained from Sigma-Aldrich led to significant different results of the intracellular Ca2+ content as well as PS exposure. To investigate this phenomenon in more detail we performed experiments with LPA from 4 different batches, 2 from Sigma-Aldrich, one from Cayman Chemical Company, and one from Santa Cruz Biotechnology Inc. All 4 LPA probes were freshly ordered, re-frozen only once and were applied to the same blood on the same day and using the same HPS. In addition, all experiments were carried out by the same investigator using identical experimental conditions (see below). The results for double labelling measurement of intracellular Ca2+ content (with x-rhod-1) and PS exposure (with annexin V-FITC) in RBCs at LPA incubation times of 1 min, 15 min and 30 min using flow cytometry are shown in Fig. 1. The control data, i.e. Ca2+ content and PS exposure of RBCs without LPA activation, are shown at time zero. In all cases of LPA activation there is a significant increase in the percentage of RBCs showing increased intracellular Ca2+ content (Fig. 1A) and PS exposure (Fig. 1B) compared to control. Interestingly, there are significant differences in the percentages of RBCs responding with increased intracellular Ca2+ content as well as PS exposure depending on the LPA batch used. The highest LPA activation was obtained using one batch from Sigma-Aldrich (batch 1) and from Cayman Chemical Company (not significantly different) followed by a batch from Santa Cruz Biotechnology Inc. (significantly different from the two batches mentioned before). LPA from the second batch from Sigma-Aldrich (batch 2) resulted in much less stimulation (significantly different from all others) at any time point measured. In addition, we found that the LPA activation efficiency within a single batch decreased with the number of times the LPA stock solution was frozen and thawed. After 3 times thawing, the activation efficiency for Ca2+ measurements decreased by about 37% and 26% for fluo-4 and x-rhod-1, respectively. At the same time the activation efficiency for PS exposure decreased by 55%, both for annexin V-FITC and annexin V alexa fluor® 647. The findings are of importance comparing results of RBC activation with LPA from different batches of Sigma-Aldrich as well as from different companies. For our investigations reported in the present paper we divided the obtained LPA into aliquots to avoid repeated thawing.

Fig. 1

Percentage of RBCs (A) responding with increased intracellular Ca2+ content (measured using x-rhod-1) and (B) responding with increased PS exposure (measured using annexin V-FITC) after activation with LPA (2.5 µM) obtained from different companies depending on time using flow cytometry. White square: LPA from Sigma-Aldrich (batch 1); dark square: LPA from Sigma-Aldrich (batch 2); dark circle: LPA from Cayman Chemical Company; dark triangle: LPA from Santa Cruz Biotechnology Inc. N = 3 (90,000 cells), error bars = S.D. (only half error bar is shown for convenience). Significant differences for LPA batches at a certain time, ANOVA (0.01 < p ≤ 0.05 (*); 0.001 < p ≤ 0.01 (**); p ≤ 0.001 (***)); for Ca2+ content, time point 1 min: * Cayman and Sigma 2 vs. Santa Cruz, *** Cayman and Sigma 1 vs. Sigma 2; time point 15 min: ** Santa Cruz vs. Sigma 2, *** Cayman vs. Sigma 2, Sigma 1 vs. Sigma 2; time point 30 min: ** Santa Cruz vs. Sigma 2, Sigma 1 vs. Sigma 2, *** Cayman vs. Sigma 2; for PS exposure, time point 1 min: * Cayman vs. Sigma 2; time point 15 min: ** Santa Cruz vs. Sigma 2, *** Cayman vs. Sigma 2, Sigma 1 vs. Sigma 2; time point 30 min: * Cayman vs. Santa Cruz, ** Sigma 1 vs. Sigma 2, *** Cayman vs. Sigma 2. Significant differences for LPA batches for PS exposure depending on time: LPA from Sigma 1: * 1 min vs. 30 min, ** 1 min vs. 15 min; LPA from Cayman: *** 1 min vs. 15 min and 30 min.

http://www.karger.com/WebMaterial/ShowPic/510038

Comparison of RBC activation using LPA with different mixing conditions

To study a possible effect of the experimenter on the results obtained, we performed experiments with two different ways of mixing the RBC suspensions after LPA activation. Double labelling experiments for Ca2+ content (using x-rhod-1) and PS exposure (using annexin V-FITC) after activation of RBCs with LPA obtained from Cayman Chemical Company were compared following simple shaking by hand and with vortexing the RBC suspensions (both in Eppendorf tubes, 5 s each). The results are presented in Fig. 2. One can observe a significant higher percentage of RBCs responding with increased intracellular Ca2+ content as well as PS exposure when the suspension is vortexed after addition of LPA compared with simple shaking by hand. Similar relationships were obtained when the fluorescent dye combination fluo-4 (to detect Ca2+) and annexin V alexa fluor® 647 (to detect PS) has been used (data not shown). These results suggest that a mechanosensitive channel, like the recently reported Piezo1, may contribute to the LPA-induced Ca2+ entry ([34] and references therein).

Fig. 2

Percentage of RBCs responding with increased intracellular Ca2+ content (detected with x-rhod-1) as well as PS exposure (detected with annexin V-FITC) after activation with LPA obtained from Cayman Chemical Company (2.5 µM) for 1 min with and without shaking by hand or vortexing the RBC suspensions using flow cytometry. Left 3 columns: RBCs with increased intracellular Ca2+ content. Right 3 columns: RBCs with increased PS exposure: White bars: control measurements (without LPA activation), grey bars: shaking the RBC suspensions by hand for 5 s, black bars: vortexing the RBC suspensions for 5 s. N = 3 (90,000 cells), error bars = S.D. (only half error bar is shown for convenience). Significant differences, ANOVA (0.01 < p ≤ 0.05 (*); 0.001 < p ≤ 0.01 (**); p ≤ 0.001 (***)); for Ca2+ content: ** control vs. LPA with shaking by hand, LPA with shaking by hand vs. LPA with vortexing, *** control vs. LPA with vortexing. For PS exposure: * control vs. LPA with shaking by hand, ** LPA with shaking by hand vs. LPA with vortexing, *** control vs. LPA with vortexing.

http://www.karger.com/WebMaterial/ShowPic/510037

Comparison of RBC activation using LPA between single and double labelling. Effect of different fluorescent dyes

The intracellular Ca2+ content and the PS exposure of RBCs can be measured on the basis of single as well as double labelling experiments. Furthermore, different fluorescent dyes are available for the detection of either parameter. It was shown that fura-2 cannot be used for Ca2+ measurement in RBCs because of the haemoglobin absorption [35]. Commonly used for the Ca2+ and PS measurements are fluo-4 (or fluo-3) and annexin V-FITC, respectively [36,37,38,39]. However, for double labelling experiment the combination of these dyes is not suitable since they are both derivatives of fluorescein and thus their absorption and emission spectra are similar. Therefore, we compared results obtained on the basis of single labelling (fluo-4 and x-rhod-1 for Ca2+, annexin V alexa fluor® 647 and annexin V-FITC for PS) and double labelling experiments using a combination of fluo-4 (for Ca2+) / annexin V alexa fluor® 647 (for PS) and x-rhod-1 (for Ca2+) / annexin V-FITC (for PS). The data after 1 min LPA activation are shown in Fig. 3. For these experiments LPA from Cayman Chemical Company was used. Fig. 3A indicates that for intracellular Ca2+ content there is no significant difference between single and double labelling experiments. In addition, the results do not depend on the fluorescent dye (or the combination of the fluorescent dyes) used. The values for PS exposure are illustrated in Fig. 3B. It can be seen that the values of double labelling experiments are significantly lower for both fluorescent dyes (annexin V-FITC and annexin V alexa fluor® 647 (for PS), in combination with x-rhod-1 and fluo-4 (for Ca2+), respectively) compared to single labelling experiments (annexin V-FITC or annexin V alexa fluor® 647 alone (for PS)). In addition, the amount of RBCs showing PS exposure is also significantly different in single labelling experiments when the cells were stained with annexin V-FITC in comparison to annexin V alexa fluor® 647 as well as in the corresponding double labelling experiments. Therefore, the combination of x-rhod-1 (to detect Ca2+ content) and annexin V-FITC (to detect PS exposure) seems to be more efficient than the combination of fluo-4 and annexin V alexa fluor® 647 in double labelling experiments. However, it should be taken into consideration that the PS data obtained in double labeling experiments are only hardly to compare with data obtained from single labeling measurements, because of the Ca2+ buffering by the Ca2+ fluorophor (see below).

Fig. 3

Percentage of RBCs (A) responding with increased intracellular Ca2+ content and (B) with increased PS exposure after 1 min activation with LPA (2.5 µM) obtained from Cayman Chemical Company using flow cytometry. Comparison between single labelling (SL) and double labelling (DL) experiments using x-rhod-1 and fluo-4 for Ca2+ detection and annexin V-FITC and annexin V alexa fluor® 647 for PS detection. All experiments were done with vortexing the RBC suspensions. (A): Black column: SL, fluo-4, dark grey column: DL, fluo-4 and annexin V alexa fluor® 647 (AV647), light grey column: SL, x-rhod-1, white column: DL, x-rhod-1 and annexin V-FITC (AV-FITC). (B): Black column: SL, annexin V alexa fluor® 647 (AV647), dark grey column: DL, annexin V alexa fluor® 647 (AV647) and fluo-4, light grey column: SL, annexin V-FITC (AV-FITC), white column: DL, annexin V-FITC (AV-FITC) and x-rhod-1. N = 3 (90,000 cells), error bars = S.D. (only half error bar is shown for convenience). Significant differences, ANOVA (0.01 < p ≤ 0.05 (*); 0.001 < p ≤ 0.01 (**); p ≤ 0.001 (***)) are shown in the figure.

http://www.karger.com/WebMaterial/ShowPic/510036

In another set of experiments we investigated the intracellular Ca2+ content and PS exposure of RBCs after 30 min LPA activation. For these experiments LPA from one batch obtained from Sigma-Aldrich (batch 1) has been used. Results of single and double labelling experiments using different fluorescent dyes can be seen in Fig. 4. Figure 4A and 4B present data of Ca2+ and PS measurements, respectively, in which on the left side flow cytometry data and on the right side data obtained using fluorescence microscopy are shown (for analysing fluorescence microscopy images see below).

Fig. 4

Percentage of RBCs (A) responding with increased intracellular Ca2+ content and (B) with increased PS exposure after 30 min activation with LPA (2.5 µM) obtained from Sigma-Aldrich (batch 1). Comparison between flow cytometry (left 4 columns) and fluorescence microscopy (right 4 columns) measurements based on single labelling (SL) and double labelling (DL) experiments using x-rhod-1 and fluo-4 for Ca2+ detection and annexin V-FITC and annexin V alexa fluor® 647 for PS detection. All experiments were done with vortexing the RBC suspensions. (A): Black column: SL, fluo-4, dark grey column: DL, fluo-4 and annexin V alexa fluor® 647 (AV647), light grey column: SL, x-rhod-1, white column: DL, x-rhod-1 and annexin V-FITC (AV-FITC). (B): Black column: SL, annexin V alexa fluor® 647 (AV647), dark grey column: DL, annexin V alexa fluor® 647 (AV647) and fluo-4, light grey column: SL, annexin V-FITC (AV-FITC), white column: DL, annexin V-FITC (AV-FITC) and x-rhod-1. N = 3 (90,000 cells), error bars = S.D. (only half error bar is shown for convenience). Significant differences, ANOVA (0.01 < p ≤ 0.05 (*); 0.001 < p ≤ 0.01 (**); p ≤ 0.001 (***)) are shown in the figure.

http://www.karger.com/WebMaterial/ShowPic/510035

Similar to the results obtained after 1 min LPA activation, there are no significant differences in the Ca2+ content measured in single labelling experiments (fluo-4 and x-rhod-1 for Ca2+) and the corresponding double labelling experiments using a combination of fluo-4 (for Ca2+) / annexin V alexa fluor® 647 (for PS) and x-rhod-1 (for Ca2+) / annexin V-FITC (for PS). In addition, the results of the experiments using fluo-4 and x-rhod-1 are also not significantly different (Fig. 4A, left). Comparing the data after 1 min and 30 min LPA activation (Figs. 3A and 4A), one can see in all cases a decrease of the Ca2+ content after 30 min. Such behaviour has been reported and explained in a recent publication [16]. For PS exposure, nearly the same tendency after 30 min compared to 1 min LPA activation can be seen (Fig. 4B, left). The results of single as well as double labelling experiment using annexin V alexa fluor® 647 for PS detection are again lower compared to the experiments where annexin V-FITC has been used (but only the double labelling data are significantly different). In addition, as for the 1 min LPA activation experiments, the amount of RBCs showing PS exposure is also significantly lower in double labelling compared to single labelling experiments when the cells were stained with annexin V alexa fluor® 647. Comparing the data after 1 min and 30 min LPA activation (Figs. 3B and 4B), one can see in all cases of measurement an increase of the PS exposure after 30 min. Such behaviour has been reported and explained in a recent publication [16].

The reasons for the differences in single labelling versus double labelling experiments as presented in Figs. 3 and 4 are multifactorial and can be explained by three major effects: (i) the Ca2+ buffering capacities of the Ca2+ fluorophores, (ii) the properties of the fluorescent dyes, and (iii) the temporal development of the Ca2+ signals.

(i) Fluo-4 and x-rhod-1 have in vitro dissociation constants Kd for Ca2+ of 345 nM and 700 nM, respectively [40]. In living cells these Kd's are, dependent on their cellular localisation, and are usually increased [41]. Especially when taking the low resting Ca2+ concentration in RBCs of around 60 nM [42] into account, the buffering capacity of the Ca2+ fluorophors loaded into the cells is high. The variety of the cellular responses [21] may add to the observed effects. Although the EC50 of the scramblase (30-70 µM) compared to the in vivo Kd of fluo-4 (1 µM) [43] is several fold higher, there are observations suggesting that scrambling may actually require much less Ca2+[44], and therefore the buffering of the Ca2+ is the most likely explanation for a lower PS exposure in the double labelled cells as depicted in Figs. 3B and 4B. The relative decrease in PS exposure of the double labelling compared the single labelling is much higher for fluo-4 compared to x-rhod-1 (Fig. 3B). This observation is also in agreement with the Kd´s of the two fluorophors (see above) and hence their buffering properties.

(ii) Other aspects that may contribute to the differences between FITC and alexa fluor® 647 results are the photophysical properties of the dyes. Assuming that the fluorescence quantum yield of a cyanine dye (alexa fluor® 647) may change when the surrounding conditions are changed, e.g. in RBC haemoglobin close to the plasma membrane could be a factor influencing alexa fluor® 647. Isomerisation of double bonds of cyanine dyes is well known [45,46]. Such changes in combination with wavelength dependencies of the detectors may account, at least partly, for the differences observed for PS detection based on FITC and alexa fluor® 647. Differences in incubation times for single and double labelling experiments as well as the solvent DMSO can be ruled out as sources for different results (we tested this by incubating the RBCs for 30 min in DMSO but without fluo-4, data not shown).

(iii) Apart from the different LPA batches, measurements between Fig. 3 and Fig. 4 differ in their measurement time after LPA stimulation (1 min versus 30 min, respectively). After 30 min LPA stimulation, the PS exposure, which is a cumulative process, reaches in the presence of x-rhod-1 the same value as that in the absence of x-rhod-1. As mentioned before, due to its Kd, x-rhod-1 buffers the Ca2+ at a higher level compared to fluo-4. This Ca2+ level allows a basal scramblase activity, which in combination with the temporal aspects explains the differences observed between Fig. 3B and Fig. 4B.

Furthermore, it is worthwhile to mention that de-esterification of the Ca2+ fluorophores may generate formaldehyde and thus affect RBC behaviour, namely by ATP-depletion [47], which can be prevented by the addition of pyruvate [48].

Comparison of RBC activation using LPA between flow cytometry and fluorescence microscopy measurements

In addition to flow cytometry investigations of intracellular Ca2+ content and PS exposure of RBCs, the same parameters were estimated from RBCs images of the same samples using fluorescence microscopy. The results obtained are presented for comparison in Fig. 4A and 4B (right). The percentage of RBCs after LPA activation showing an evaluated intracellular Ca2+ content is slightly lower when measured using fluorescence microscopy. This is in contrast to previous investigations, where a loss of high Ca2+ cells in flow cytometry due to their increased fragility was reported [49]. However, considering the hypothesis mentioned above, that a mechanosensitive channel contributes to the LPA-induced Ca2+ entry, the shear stress and pressure in flow cytometers could explain a higher Ca2+ content in flow cytometry compared to microscopy [50,51]. Although there is such a tendency for all conditions measured, only for fluo-4 and single labelling experiments the difference is significant. For PS exposure no differences between flow cytometry and fluorescence microscopy measurements can be seen.

Conclusion

We were able to show that the results of measurements of the Ca2+ content as well as PS exposure of RBCs after LPA activation significantly depend on the LPA batches most probably due to the substance stability. We propose to handle that issue by limiting quantitative comparisons of data to those obtained with the same LPA batch. Nevertheless qualitative comparisons cross LPA batches and cross laboratories should still be possible.

We propose that differences in results due to the method of mixing LPA with the RBC suspension are caused by mechanosensitive ion channels. This hypothesis requires further investigations.

In addition, we demonstrated that annexin V-FITC is more sensitive in comparison to annexin V alexa fluor® 647 for the quantification of PS exposure both in single as well as double labelling experiments.

Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments, which is most probably caused by the Ca2+ buffering capacities of the Ca2+ indicators. It is an example how the measurement itself disturbs the process being investigated. It is an almost unavoidable side effect but should be taken into consideration.

Finally, data of Ca2+ content are slightly affected by the measuring method (flow cytometry versus fluorescence microscopy). Although compared to the issues discussed above, these are minor differences.

Acknowledgements

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106-YS.06-2013.16 for D. B. Nguyen, a grant from CNPq program “science without borders” to M. C. Wesseling, process number: 202426/2012-2 (Brasil) and from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No 602121 (CoMMiTMenT) and the EU Framework Programme for Research and Innovation Horizon 2020, Maria Curie Innovative Training Network project under grant agreement No 675115 (RELEVANCE) to L. Kaestner. The authors are grateful for the discussion of the differences of the fluorescence intensities comparing the single and double labelling experiments to Prof. Gregor Jung, Department of Physical Chemistry, Saarland University. The authors thank Jörg Riedel for technical support.

Disclosure Statement

The authors declare no conflict of interest.


References

  1. Kaestner L, Bollensdorff C, Bernhardt I: Non-selective voltage-activated cation channel in the human red blood cell membrane. Biochim Biophys Acta 1999;1417:9-15.
  2. Kaestner L, Tabellion W, Lipp P, Bernhardt I: Prostaglandin E2 activates channel-mediated calcium entry in human erythrocytes: an indicator for a blood clot formation supporting process. Thromb Haemost 2004;92:1269-1272.
  3. Wagner-Britz L, Wang J, Kaestner L, Bernhardt I: Protein kinase Cα and P-type Ca2+ channel CaV2.1 in red blood cell calcium signalling. Cell Physiol Biochem 2013;31:883-891.
  4. Lang F, Qadri SM: Mechanisms and significance of eryptosis, the suicidal death of erythrocytes. Blood Purif 2012;33:125-130.
  5. Föller M, Huber SM, Lang F: Critical Review. Erythrocyte programmed cell death. IUBMB Life 2008;60:661-668.
  6. Woon LA, Holland JW, Kable EP, Roufogalis BD: Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts. Cell Calcium 1999;25:313-320.
  7. Steffen P, Jung A, Nguyen DB, Müller T, Bernhardt I, Kaestner L, Wagner C: Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion. Cell Calcium 2011;50:54-61.
  8. Kaestner L, Steffen P, Nguyen DB, Wang J, Wagner-Britz L, Jung A, Wagner C, Bernhardt I: Lysophosphatidic acid induced red blood cell aggregation in vitro. Bioelectrochemistry 2012;87:89-95.
  9. Closse C, Dachary-Progent J, Boisseau MR: Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium. Br J Haematol 1999;107:300-302.
  10. De Jong K, Larkin SK, Styles LA, Bookchin RM, Kuypers FA: Characterization of the phosphatidylserine-exposing subpopulation of sickle cells. Blood 2001;98:860-867.
  11. Sherman IW, Prudhomme J, Tait JF: Altered membrane phospholipid asymmetry in plasmodium falciparum-infected erythrocytes. Parasitol Today 1997;13:242-243.
  12. Wali RK, Jaffe S, Kumar D, Kalra VK: Alterations in organization of phospholipids in erythrocytes as factor in adherence to endothelial cells in diabetes mellitus. Diabetes 1988;37:104-111.
  13. Andrews D, Low PS: Role of red blood cells in thrombosis. Curr Opin Hematol 1999;6:76-82.
  14. Hellem AJ: The adhesiveness of human blood platelets in vitro. Scand J Clin Lab Invest 1960;12:1-117.
  15. Nguyen DB, Wagner-Britz L, Maia S, Steffen P, Wagner C, Kaestner L, Bernhardt I: Regulation of phosphatidylserine exposure in red blood cells. Cell Physiol Biochem 2011;28:847-856.
  16. Wesseling MC, Wagner-Britz L, Huppert H, Hanf B, Hertz L, Nguyen DB, Bernhardt I: Phosphatidylserine exposure in human red blood cells depending on cell age. Cell Physiol Biochem 2016;38:1376-1390.
  17. Andrews DA, Yang L, Low PS: Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells. Blood 2002;100:3392-3399.
  18. De Jong K, Retting MP, Low PS, Kuypers FA: Protein kinase C activation induces phosphatidylserine exposure on red blood cells. Biochem 2002;41:12562-12567.
  19. Tang F, Ren Y, Wang R, Lei X, Deng X, Zhao Y, Chen D, Wang X: Ankyrin exposure induced by activated protein kinase C plays a potential role in erythrophagocytosis. Biochim Biophys Acta 2016;1860:120-128.
  20. Neidlinger NA, Larkin SK, Bhagat A, Victorino GP, Kuypers FA: Hydrolysis of phosphatidylserine-exposing red blood cells by secretory phospholipase A2 generates lysophosphatidic acid and results in vascular dysfunction. J Biol Chem 2006;281:775-781.
  21. Wang J, Wagner-Britz L, Bogdanova A, Ruppenthal S, Wiesen K, Kaiser E, Tian Q, Krause E, Bernhardt I, Lipp P, Philipp SE, Kaestner L: Morphologically homogeneous red blood cells present a heterogeneous response to hormonal stimulation. PLoS ONE 2013;8:e67697.
  22. Siegl C, Hamminger P, Jank H, Ahting U, Bader B, Danek A, Gregory A, Hartig M, Hayflick S, Hermann A, Prokisch H, Sammler EM, Yapici Z, Phrohaska R, Salzer U: Alterations of red cell membrane properties in Nneuroacanthocytosis. PLoS ONE 2013;8:e76715.
  23. Chung SM, Bae ON, Lim KM, Noh JY, Lee MY, Jung YS, Chung JH: Lysophosphatidic acid induces thrombogenic activity through phosphatidylserine exposure and procoagulant microvesicle generation in human erythrocytes. Arterisoscler Thromb Vasc Biol 2007;27:414-421.
  24. Lang KS, Lang PA, Bauer C, Duranton C, Wieder T, Huber SM, Lang F: Mechanisms of suicidal erythrocyte death. Cell Physiol Biochem 2005;15:195-202.
  25. Peter T, Bissinger R, Enkel S, Alzoubi K, Oswald G, Lang F: Programmed erythrocyte death following in vitro Treosulfan treatment. Cell Physiol Biochem 2015;35:1372-80.
  26. Faggio C, Alzoubi K, Calabrò S, Lang F: Stimulation of suicidal erythrocyte death by PRIMA-1. Cell Physiol Biochem 2015;35:529-40.
  27. Zhang R, Xiang Y, Ran Q, Deng X, Xiao Y, Xiang L, Li Z: Involvement of calcium, reactive oxygen species, and ATP in hexavalent chromium-induced damage in red blood cells. Cell Physiol Biochem 2014;34:1780-91.
  28. Arnold M, Bissinger R, Lang F: Mitoxantrone-induced suicidal erythrocyte death. Cell Physiol Biochem 2014;34:1756-67.
  29. Tesoriere L, Attanzio A, Allegra M, Cilla A, Gentile C, Livrea MA: Oxysterol mixture in hypercholesterolemia-relevant proportion causes oxidative stress-dependent eryptosis. Cell Physiol Biochem 2014;34:1075-89.
  30. Lupescu A, Bissinger R, Warsi J, Jilani K, Lang F: Stimulation of erythrocyte cell membrane scrambling by gedunin. Cell Physiol Biochem 2014;33:1838-48.
  31. Jacobi J, Lang E, Bissinger R, Frauenfeld L, Modicano P, Faggio C, Abed M, Lang F: Stimulation of erythrocyte cell membrane scrambling by mitotane. Cell Physiol Biochem 2014;33:1516-26.
  32. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995;184:39-51.
  33. Kucherenko YV, Bernhardt I: Natural antioxidants improve red blood cell "survival" in non-leukoreduced blood samples. Cell Physiol Biochem 2015;35:2055-68.
  34. Kaestner L: Channelizing the red blood cell: molecular biology competes with patch-clamp. Front Mol Biosci 2015;2:46.
  35. Kaestner L, Tabellion W, Weiss E, Bernhardt I, Lipp P: Calcium imaging of individual erythrocytes: Problems and approaches. Cell Calcium 2006;39:13-19.
  36. Ghashghaeinia M, Cluitmans JC, Akel A, Dreischer P, Toulany M, Köberle M, Skabytska Y, Saki M, Biedermann T, Duszenko M, Lang F, Wieder T, Bosman GJ: The impact of erythrocyte age on eryptosis. Br J Haematol 2012;157:606-614.
  37. Ghashghaeinia M, Cluitmans JC, Toulany M, Saki M, Köberle M, Lang E, Dreischer P, Biedermann T, Duszenko M, Lang F, Bosman GJ, Wieder T: Age sensitivity of NFκB abundance and programmed cell death in erythrocytes induced by NFκB inhibitors. Cell Physiol Biochem 2013;32:801-813.
  38. Lang E, Bissinger R, Fajol A, Salker MS, Singh J, Zelenak C, Ghashghaeinia M, Gu S, Jilani K, Lupescu A, Reyskens KMSE, Ackermann TF, Föller M, Schleicher E, Sheffield WP, Arthur JSC, Lang F, Qadri SM: Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2. Sci Reports 2015;5:17316.
  39. Klarl BA, Lang PA, Kempe DS, Niemoeller OM, Akel A, Sobiesiak M, Eisele K, Podolski M, Huber SM, Wieder T, Lang F: Protein kinase C mediates erythrocyte “programmed cell death” following glucose depletion. Am J Physiol Cell Physiol 2006;290:C244-253.
  40. Haugeland RP: Handbook of fluorescent probes and research products, ed 9. Molecular Probes Inc, 2002.
  41. Lipp P, Kaestner L: Detecting calcium in cardiac muscle: fluorescence to dye for. Am J Physiol Heart Circ Physiol 2014;307:H1687-1690.
  42. Tiffert T, Bookchin RM, Lew VL: Calcium homeostasis in normal and abnormal human red cells; In Bernhardt I, Ellory JC (eds): Red cell membrane transport in health and disease. Heidelberg, Springer Verlag, 2003, pp 373-405.
  43. Bogdanova A, Makhro A, Wang J, Lipp P, Kaestner L: Calcium in red blood cells - a perilous balance. Int J Mol Sci 2013;14:9848-9872.
  44. Weiss E, Cytlak UM, Rees DC, Osei A, Gibson JS: Deoxygenation-induced and Ca2+ dependent phosphatidylserine externalisation in red blood cells from normal individuals and sickle cell patients. Cell Calcium 2012;51:51-56.
  45. Widengren J, Schwille P: Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy. J Phys Chem 2000;104:6416-6428.
  46. Zanetti-Domingues LC, Tynan CJ, Rolfe DJ, Clarke DT, Martin-Fernandez M: Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding. PLoS ONE 2013;8:e74200.
  47. Tiffert T, García-Sancho J, Lew VL: Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells. Biochim Biophys Acta 1984;773:143-156.
  48. García-Sancho J: Pyruvate prevents the ATP depletion caused by formaldehyde or calcium-chelator esters in the human red cell. Biochim Biophys Acta 1985;813:148-150.
  49. Minetti G, Egée S, Mörsdorf D, Steffen P, Makhro A, Achilli C, Ciana A, Wang J, Bouyer G, Bernhardt I, Wagner C, Thomas S, Bogdanova A, Kaestner L: Red cell investigations: art and artefacts. 2013;27:91-101.
  50. Bouchy M, Donner M, Andre JC: Erythrocyte membranes alteration in a shear stress measured by fluorescence anisotropy. Cell Biophys 1990;17:213-225.
  51. Lakowicz JR: Principles of fluorescence spectroscopy, ed 3. New York, Springer Science + Business Media, LLC, 2006.

Author Contacts

Prof. Dr. Ingolf Bernhardt

Laboratory of Biophysics, Faculty of Natural and Technical Sciences III, Saarland

University, Campus, 66123 Saarbrücken, (Germany)

Tel. +49 681 3026689, Fax +49 681 3026690, E-Mail i.bernhardt@mx.uni-saarland.de


Article / Publication Details

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Abstract of Original Paper

Accepted: May 17, 2016
Published online: June 13, 2016
Issue release date: June 2016

Number of Print Pages: 12
Number of Figures: 4
Number of Tables: 0

ISSN: 1015-8987 (Print)
eISSN: 1421-9778 (Online)

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References

  1. Kaestner L, Bollensdorff C, Bernhardt I: Non-selective voltage-activated cation channel in the human red blood cell membrane. Biochim Biophys Acta 1999;1417:9-15.
  2. Kaestner L, Tabellion W, Lipp P, Bernhardt I: Prostaglandin E2 activates channel-mediated calcium entry in human erythrocytes: an indicator for a blood clot formation supporting process. Thromb Haemost 2004;92:1269-1272.
  3. Wagner-Britz L, Wang J, Kaestner L, Bernhardt I: Protein kinase Cα and P-type Ca2+ channel CaV2.1 in red blood cell calcium signalling. Cell Physiol Biochem 2013;31:883-891.
  4. Lang F, Qadri SM: Mechanisms and significance of eryptosis, the suicidal death of erythrocytes. Blood Purif 2012;33:125-130.
  5. Föller M, Huber SM, Lang F: Critical Review. Erythrocyte programmed cell death. IUBMB Life 2008;60:661-668.
  6. Woon LA, Holland JW, Kable EP, Roufogalis BD: Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts. Cell Calcium 1999;25:313-320.
  7. Steffen P, Jung A, Nguyen DB, Müller T, Bernhardt I, Kaestner L, Wagner C: Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion. Cell Calcium 2011;50:54-61.
  8. Kaestner L, Steffen P, Nguyen DB, Wang J, Wagner-Britz L, Jung A, Wagner C, Bernhardt I: Lysophosphatidic acid induced red blood cell aggregation in vitro. Bioelectrochemistry 2012;87:89-95.
  9. Closse C, Dachary-Progent J, Boisseau MR: Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium. Br J Haematol 1999;107:300-302.
  10. De Jong K, Larkin SK, Styles LA, Bookchin RM, Kuypers FA: Characterization of the phosphatidylserine-exposing subpopulation of sickle cells. Blood 2001;98:860-867.
  11. Sherman IW, Prudhomme J, Tait JF: Altered membrane phospholipid asymmetry in plasmodium falciparum-infected erythrocytes. Parasitol Today 1997;13:242-243.
  12. Wali RK, Jaffe S, Kumar D, Kalra VK: Alterations in organization of phospholipids in erythrocytes as factor in adherence to endothelial cells in diabetes mellitus. Diabetes 1988;37:104-111.
  13. Andrews D, Low PS: Role of red blood cells in thrombosis. Curr Opin Hematol 1999;6:76-82.
  14. Hellem AJ: The adhesiveness of human blood platelets in vitro. Scand J Clin Lab Invest 1960;12:1-117.
  15. Nguyen DB, Wagner-Britz L, Maia S, Steffen P, Wagner C, Kaestner L, Bernhardt I: Regulation of phosphatidylserine exposure in red blood cells. Cell Physiol Biochem 2011;28:847-856.
  16. Wesseling MC, Wagner-Britz L, Huppert H, Hanf B, Hertz L, Nguyen DB, Bernhardt I: Phosphatidylserine exposure in human red blood cells depending on cell age. Cell Physiol Biochem 2016;38:1376-1390.
  17. Andrews DA, Yang L, Low PS: Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells. Blood 2002;100:3392-3399.
  18. De Jong K, Retting MP, Low PS, Kuypers FA: Protein kinase C activation induces phosphatidylserine exposure on red blood cells. Biochem 2002;41:12562-12567.
  19. Tang F, Ren Y, Wang R, Lei X, Deng X, Zhao Y, Chen D, Wang X: Ankyrin exposure induced by activated protein kinase C plays a potential role in erythrophagocytosis. Biochim Biophys Acta 2016;1860:120-128.
  20. Neidlinger NA, Larkin SK, Bhagat A, Victorino GP, Kuypers FA: Hydrolysis of phosphatidylserine-exposing red blood cells by secretory phospholipase A2 generates lysophosphatidic acid and results in vascular dysfunction. J Biol Chem 2006;281:775-781.
  21. Wang J, Wagner-Britz L, Bogdanova A, Ruppenthal S, Wiesen K, Kaiser E, Tian Q, Krause E, Bernhardt I, Lipp P, Philipp SE, Kaestner L: Morphologically homogeneous red blood cells present a heterogeneous response to hormonal stimulation. PLoS ONE 2013;8:e67697.
  22. Siegl C, Hamminger P, Jank H, Ahting U, Bader B, Danek A, Gregory A, Hartig M, Hayflick S, Hermann A, Prokisch H, Sammler EM, Yapici Z, Phrohaska R, Salzer U: Alterations of red cell membrane properties in Nneuroacanthocytosis. PLoS ONE 2013;8:e76715.
  23. Chung SM, Bae ON, Lim KM, Noh JY, Lee MY, Jung YS, Chung JH: Lysophosphatidic acid induces thrombogenic activity through phosphatidylserine exposure and procoagulant microvesicle generation in human erythrocytes. Arterisoscler Thromb Vasc Biol 2007;27:414-421.
  24. Lang KS, Lang PA, Bauer C, Duranton C, Wieder T, Huber SM, Lang F: Mechanisms of suicidal erythrocyte death. Cell Physiol Biochem 2005;15:195-202.
  25. Peter T, Bissinger R, Enkel S, Alzoubi K, Oswald G, Lang F: Programmed erythrocyte death following in vitro Treosulfan treatment. Cell Physiol Biochem 2015;35:1372-80.
  26. Faggio C, Alzoubi K, Calabrò S, Lang F: Stimulation of suicidal erythrocyte death by PRIMA-1. Cell Physiol Biochem 2015;35:529-40.
  27. Zhang R, Xiang Y, Ran Q, Deng X, Xiao Y, Xiang L, Li Z: Involvement of calcium, reactive oxygen species, and ATP in hexavalent chromium-induced damage in red blood cells. Cell Physiol Biochem 2014;34:1780-91.
  28. Arnold M, Bissinger R, Lang F: Mitoxantrone-induced suicidal erythrocyte death. Cell Physiol Biochem 2014;34:1756-67.
  29. Tesoriere L, Attanzio A, Allegra M, Cilla A, Gentile C, Livrea MA: Oxysterol mixture in hypercholesterolemia-relevant proportion causes oxidative stress-dependent eryptosis. Cell Physiol Biochem 2014;34:1075-89.
  30. Lupescu A, Bissinger R, Warsi J, Jilani K, Lang F: Stimulation of erythrocyte cell membrane scrambling by gedunin. Cell Physiol Biochem 2014;33:1838-48.
  31. Jacobi J, Lang E, Bissinger R, Frauenfeld L, Modicano P, Faggio C, Abed M, Lang F: Stimulation of erythrocyte cell membrane scrambling by mitotane. Cell Physiol Biochem 2014;33:1516-26.
  32. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995;184:39-51.
  33. Kucherenko YV, Bernhardt I: Natural antioxidants improve red blood cell "survival" in non-leukoreduced blood samples. Cell Physiol Biochem 2015;35:2055-68.
  34. Kaestner L: Channelizing the red blood cell: molecular biology competes with patch-clamp. Front Mol Biosci 2015;2:46.
  35. Kaestner L, Tabellion W, Weiss E, Bernhardt I, Lipp P: Calcium imaging of individual erythrocytes: Problems and approaches. Cell Calcium 2006;39:13-19.
  36. Ghashghaeinia M, Cluitmans JC, Akel A, Dreischer P, Toulany M, Köberle M, Skabytska Y, Saki M, Biedermann T, Duszenko M, Lang F, Wieder T, Bosman GJ: The impact of erythrocyte age on eryptosis. Br J Haematol 2012;157:606-614.
  37. Ghashghaeinia M, Cluitmans JC, Toulany M, Saki M, Köberle M, Lang E, Dreischer P, Biedermann T, Duszenko M, Lang F, Bosman GJ, Wieder T: Age sensitivity of NFκB abundance and programmed cell death in erythrocytes induced by NFκB inhibitors. Cell Physiol Biochem 2013;32:801-813.
  38. Lang E, Bissinger R, Fajol A, Salker MS, Singh J, Zelenak C, Ghashghaeinia M, Gu S, Jilani K, Lupescu A, Reyskens KMSE, Ackermann TF, Föller M, Schleicher E, Sheffield WP, Arthur JSC, Lang F, Qadri SM: Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2. Sci Reports 2015;5:17316.
  39. Klarl BA, Lang PA, Kempe DS, Niemoeller OM, Akel A, Sobiesiak M, Eisele K, Podolski M, Huber SM, Wieder T, Lang F: Protein kinase C mediates erythrocyte “programmed cell death” following glucose depletion. Am J Physiol Cell Physiol 2006;290:C244-253.
  40. Haugeland RP: Handbook of fluorescent probes and research products, ed 9. Molecular Probes Inc, 2002.
  41. Lipp P, Kaestner L: Detecting calcium in cardiac muscle: fluorescence to dye for. Am J Physiol Heart Circ Physiol 2014;307:H1687-1690.
  42. Tiffert T, Bookchin RM, Lew VL: Calcium homeostasis in normal and abnormal human red cells; In Bernhardt I, Ellory JC (eds): Red cell membrane transport in health and disease. Heidelberg, Springer Verlag, 2003, pp 373-405.
  43. Bogdanova A, Makhro A, Wang J, Lipp P, Kaestner L: Calcium in red blood cells - a perilous balance. Int J Mol Sci 2013;14:9848-9872.
  44. Weiss E, Cytlak UM, Rees DC, Osei A, Gibson JS: Deoxygenation-induced and Ca2+ dependent phosphatidylserine externalisation in red blood cells from normal individuals and sickle cell patients. Cell Calcium 2012;51:51-56.
  45. Widengren J, Schwille P: Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy. J Phys Chem 2000;104:6416-6428.
  46. Zanetti-Domingues LC, Tynan CJ, Rolfe DJ, Clarke DT, Martin-Fernandez M: Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding. PLoS ONE 2013;8:e74200.
  47. Tiffert T, García-Sancho J, Lew VL: Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells. Biochim Biophys Acta 1984;773:143-156.
  48. García-Sancho J: Pyruvate prevents the ATP depletion caused by formaldehyde or calcium-chelator esters in the human red cell. Biochim Biophys Acta 1985;813:148-150.
  49. Minetti G, Egée S, Mörsdorf D, Steffen P, Makhro A, Achilli C, Ciana A, Wang J, Bouyer G, Bernhardt I, Wagner C, Thomas S, Bogdanova A, Kaestner L: Red cell investigations: art and artefacts. 2013;27:91-101.
  50. Bouchy M, Donner M, Andre JC: Erythrocyte membranes alteration in a shear stress measured by fluorescence anisotropy. Cell Biophys 1990;17:213-225.
  51. Lakowicz JR: Principles of fluorescence spectroscopy, ed 3. New York, Springer Science + Business Media, LLC, 2006.
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